Vascular endothelial growth factor levels in tuberculosis: A systematic review and meta-analysis

Background Changes in endothelial function are implicated in the spread of tuberculosis (TB). Studies suggest a role for the vascular endothelial growth factor (VEGF) in TB-related endothelial function changes. However, the findings of studies investigating the VGEF profile in TB are not consistent, and no formal systematic review and meta-analysis exists summarizing these studies. Methods We did a meta-analysis of studies assessing VEGF levels in patients with TB. A systematic search on June 25, 2021, was conducted for eligible studies that made VEGF measurements in an unstimulated sample, e.g., a blood fraction (plasma or serum), cerebrospinal fluid (CSF), pleural effusion (PE), or bronchoalveolar lavage fluid, and ascites or pericardial fluid for patients with TB and controls without TB. Also, studies that made simultaneous measurements of VEGF in blood and PE or CSF in the same patients with TB were included. Longitudinal studies that provided these data at baseline or compared pre-post anti-tuberculosis treatment (ATT) levels of VEGF were included. The primary outcome was the standardized mean difference (SMD) of VEGF levels between the comparison groups. Results 52 studies were included in the meta-analysis. There were 1787 patients with TB and 3352 control subjects of eight categories: 107 patients with transudative pleural effusion, 228 patients with congestive heart failure (CHF)/chronic renal failure (CRF), 261 patients with empyema and parapneumonic effusion (PPE), 241 patients with cirrhosis, 694 healthy controls (with latent TB infection or uninfected individuals), 20 patients with inactive tuberculous meningitis (TBM), 123 patients with non-TBM, and 1678 patients with malignancy. The main findings are as follows: (1) serum levels of VEGF are higher in patients with active TB compared with healthy controls without other respiratory diseases, including those with latent TB infection or uninfected individuals; (2) both serum and pleural levels of VEGF are increased in patients with TPE compared with patients with transudative, CHF/CRF, or cirrhotic pleural effusion; (3) ascitic/pericardial fluid, serum, and pleural levels of VEGF are decreased in patients with TB compared with patients with malignancy; (4) pleural levels of VEGF are lower in patients with TPE compared with those with empyema and PPE, whereas serum levels of VEGF are not different between these patients; (5) both CSF and serum levels of VEGF are increased in patients with active TBM compared with controls, including patients with inactive TBM or non-TBM subjects; (6) post-ATT levels of VEGF are increased compared with pre-ATT levels of VEGF; and (7) the mean age and male percentage of the TB group explained large and total amount of heterogeneity for the meta-analysis of blood and pleural VEGF levels compared with healthy controls and patients with PPE, respectively, whereas these moderators did not show any significant interaction with the effect size for other analyses. Discussion The important limitation of the study is that we could not address the high heterogeneity among studies. There might be unmeasured factors behind this heterogeneity that need to be explored in future research. Meta-analysis findings align with the hypothesis that TB may be associated with abnormal vascular function, and both local and systemic levels of VEGF can be used to trace this abnormality.

compared with healthy controls without other respiratory diseases, including those with latent TB infection or uninfected individuals; (2) both serum and pleural levels of VEGF are increased in patients with TPE compared with patients with transudative, CHF/CRF, or cirrhotic pleural effusion; (3) ascitic/pericardial fluid, serum, and pleural levels of VEGF are decreased in patients with TB compared with patients with malignancy; (4) pleural levels of VEGF are lower in patients with TPE compared with those with empyema and PPE, whereas serum levels of VEGF are not different between these patients; (5) both CSF and serum levels of VEGF are increased in patients with active TBM compared with controls, including patients with inactive TBM or non-TBM subjects; (6) post-ATT levels of VEGF are increased compared with pre-ATT levels of VEGF; and (7) the mean age and male percentage of the TB group explained large and total amount of heterogeneity for the meta-analysis of blood and pleural VEGF levels compared with healthy controls and patients with PPE, respectively, whereas these moderators did not show any significant interaction with the effect size for other analyses.

Introduction
Despite a century of intense medical research, the burden of tuberculosis (TB) remains alarming due to its causing pathogen, mycobacterium tuberculosis (M. tb), that interferes with the biology of molecules and cells to seriously affect the function of vital organs and systems. This pathogen has been shown to invade the circulatory system, respiratory system, central nervous system, lymphatic system, gastrointestinal system, and genitourinary system. There are different, yet unproven, mechanisms proposed to handle such an invasion. However, the hypothesis of vascular involvement appears to be functional. In particular, the development of vascular complications and elevation of adhesion molecules [1] in patients with TB support this hypothesis.
Moreover, since 2000, research has shown that the protein levels of vascular endothelial growth factor (VEGF), which correlates with angiogenesis and is traditionally considered a marker of malignant situations [2], are altered in TB. However, the findings are not consistent, and no pooled analysis exists investigating the VEGF profile in patients with TB. This inconsistency might lie in different samples used for VEGF measurement, including the cerebrospinal fluid (CSF), the peripheral blood, the pleural effusion (PE), the ascites or pericardial fluid, and different conditions served as a control condition, such as apparently healthy conditions, malignancy, empyema and parapneumonic effusion (PPE), transudate effusion, etc.
Elucidating the profile of VEGF will be helpful to diagnosis of TB and its differentiation from the aforementioned clinical conditions. In addition, the decrease or increase of this growth factor might shed light on the pathogenesis of TB and therapeutic implications. This is a systematic review and meta-analysis of studies investigating the VEGF profile in patients with TB.

Methods
We have prepared the present study according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [3] (S1 Checklist).

Search strategy
We searched PubMed, Scopus, and Web of Science using the search terms: (tuberculosis OR tuberculous) AND (vascular endothelial growth factor OR VEGF). The search was conducted on June 25, 2021, and the analyses were finalized on July 20, 2021. There was no restriction applied to language or date of publication. We also searched Google Scholar for any additional studies missing from the database search.

Selection criteria
Publication type. Original studies that met the eligibility criteria were included. Study design. Observational studies that made VEGF measurements for patients with TB and controls without TB were included. Also, observational studies that made simultaneous measurements of VEGF in blood and PE or CSF in the same patients with TB were included. Longitudinal studies that provided these data at baseline or compared pre-post anti-tuberculosis treatment (ATT) levels of VEGF were included.
Participants. Studies met the inclusion criteria if they (1) enrolled patients with TB of any age group or gender; (2) assessed VEGF levels in an unstimulated sample, e.g., a blood fraction (plasma or serum), CSF, PE or bronchoalveolar lavage fluid (BALF), and ascites or pericardial fluid; (3) compared VEGF measurements in the TB group with those of a control group, e.g., healthy controls including individuals with latent TB infection or TB-uninfected individuals without other respiratory diseases or infections, patients with malignant pleural effusion (MPE), patients with malignant ascites, patients with empyema or PPE, patients with transudate, congestive heart failure (CHF)/chronic renal failure (CRF), or cirrhotic effusion, or compared blood VEGF measurements with pleural or central VEGF measurements that were obtained simultaneously from the same patients with TB, or compared pre-ATT with post-ATT levels of VGEF in the same patients with TB; and (4) provided sufficient data to calculate the mean difference of VEGF levels between the two comparison groups.

Data extraction
We used the excel spreadsheets to extract the following data from each of the included studies: the link to the study; title; first author; year of publication; the condition, mean age, male percentage, and HIV-positive rate for each of the comparison groups; ATT status for the TB group; the sample and the assay used for VEGF measurement; the scale of VEGF measurement; and the number of participants, mean, and standard deviation (SD) of VEGF measures for each of the comparison groups. When the VEGF data were not available in the article and related supplementary material, we sent the first or corresponding author an e-mail to provide us with the required data. In addition, we used any graphs that contained data and alternative measures, e.g., median, range, interquartile range, or 95% confidence interval (CI), to estimate the mean and SD.

Quality assessment
The Newcastle-Ottawa Scale (NOS) was used for assessing the quality of included studies [4]. We appraised the studies using a platform that comprised four one-star items related to the selection of the case and control groups, two one-star items (age and sex) related to the comparability of the case and control groups, one two-star item related to the outcome assessment, and one one-star item related to the statistical tests used to analyze the data. The study quality was, thus, ranged from 0 to 9 and assigned to a high, moderate, and low when the quality scores were 7-9, 4-6, and 0-3, respectively.

Outcomes
The primary outcome was the difference in VEGF levels. The samples of interest included the ascites or pericardial fluid, BALF or PE, CSF, and plasma or serum. The between-group comparison groups of interest were: (1) patients with active TB vs. healthy controls, including individuals with latent TB infection or TB-uninfected subjects, without other respiratory diseases or TB-like diseases; (2) patients with TB ascites vs. patients with malignant ascites; (3) patients with TPE vs. patients with MPE, or patients with empyema and PPE, or patients with transudate, CHF/CRF, or cirrhotic effusion; and (4) patients with active TBM vs. patients with inactive TBM or non-TBM. The within-subject analyses of interest were blood vs. pleural or central VEGF levels and pre-ATT vs. post-ATT measures of VEGF in the same patients with TB.

Data analysis
A meta-analysis was run for a comparison of interest when three or more observations were available. The standardized mean difference (SMD) with Hedges' g was chosen as the measure of the effect. The effect size was calculated using a random-effects model with a restricted maximum-likelihood (REML) and considered a large, moderate, and small effect with respect to the SMD values of 0.8, 0.5, and 0.2, respectively. The heterogeneity among the studies included in a meta-analysis was assessed using Cochrane's Q, tau-squared, and I-squared (I 2 ). Cochrane's Q test quantifies total variance and generates a p-value that determines the heterogeneity is present. Tau-squared indicates the true variance that is the between-study variance, while I 2 represents the percentage of the total variance that is due to the true variance. The degree of heterogeneity is said to be low, moderate, and high, with I 2 values of 25%, 50%, and 75%.
Eggers' test was used to examine the funnel plot asymmetry. Whenever this test was significant with a p-value of less than 0.1, we used the trim and fill method to correct the funnel plot and adjust the effect size for potential publication bias.
Sensitivity analysis using the leave-one-out method was performed to investigate the robustness of the SMD. If the influence analysis revealed that the SMD was sensitive to the effect size of a single observation, we repeated the original analysis after removing the influential observation(s).
To investigate the possible sources of heterogeneity, subgroup analyses were done using the geographic location, the assay used (ELISA vs. not ELISA), and the sample (plasma vs. serum) as predictors. Moreover, meta-regression was considered to evaluate the interaction between potential moderators of the mean age or male percentage in the TB group and the effect size of the difference in VEGF levels.
All meta-analyses, subgroup analyses, and meta-regressions were conducted using the R (version 4.0.5).

Study characteristics
The systematic search identified 123 articles as potentially eligible to be included in our review (Fig 1), of which 74 studies were excluded with the reasons described in S1 Text. Three additional records were found by searching other sources. Finally, 52 studies were included in the meta-analysis . S1 Table summarizes

Publication bias
There was evidence of potential publication bias for meta-analyses of difference in blood VEGF levels between patients with TPE and MPE (p = 0.023) and of the difference in pleural VEGF levels between patients with TPE and MPE (p = 0.088), TPE and PPE (p = 0.080), and TPE and transudative effusions (p = 0.071). The trim and fill method changed the significance of the difference in blood VEGF levels between patients with TPE and MPE (S6 Fig). With three added studies, it was found to be not significantly different (SMD = -0.44; 95% CI, -1.71 to 0.82; p = 0.4927). Moreover, for the difference in pleural VEGF levels between patients with  The trim and fill method did not change the significance of the effect for the difference in pleural VEGF levels between patients with TPE and MPE and between patients with TPE and patients with transudative effusions or effusions due to CHF, CRF, and cirrhosis and the effect sizes remained significantly different since no additional studies needed to be imputed into the analyses.

Sensitivity analysis
The leave-one-out sensitivity analyses showed that the results of analyses of VEGF levels in the blood for patients with TB compared with healthy controls and for patients with TPE compared with MPE remained significantly robust with SMDs ranging from 1.03 to 1.  44 (S13 Fig). However, the leave-one-out method revealed the SMD of difference in pleural VEGF levels between TPE and PPE to be significant after removing an influential study by Momi and colleagues (SMD = -0.52; 95% CI, -0.90 to -0.15; I 2 = 55%; p = 0.0067) (S14 Fig and Fig 11).

Subgroup analysis
Subgroup analyses revealed that higher VEGF levels in pleural effusion than serum in patients with TB were found in studies of East Asia & Pacific (p = 0.0048), not in studies of Europe &

Meta-regression
When compared with MPE, the mean age of the TB group significantly explained a large amount of heterogeneity (R 2 = 70.5%) for blood VEGF levels (k = 6, estimated index 0.0770; p = 0.0047). In addition, the male percentage of the TB group was accounted for the total amount of heterogeneity (R 2 = 100%) for the analysis of pleural VEGF levels in TPE compared with PPE with no residual heterogeneity (p = 0.7421). There was no association between the male percentage or mean age and the effect size for other analyses (S4 Table).

Discussion
This is the first meta-analysis comparing VEGF levels between patients with TB and different control conditions, including healthy controls, patients with malignancy, patients with empyema and PPE, and patients with transudate effusion. We run meta-analyses, subgroup metaanalyses, and meta-regressions while considering different samples that could be used for VEGF measurement and other factors that might affect VEGF profile, e.g., geographic location, age, and gender. The main findings are as follows: (1) serum levels of VEGF are higher in patients with active TB compared with healthy controls without other respiratory diseases, including those with latent TB infection or uninfected individuals, whereas plasma levels of VEGF in patients with TB are not different from those of healthy controls; (2) both serum and pleural levels of VEGF are increased in patients with TPE compared with patients with transudate, CHF/CRF, or cirrhotic pleural effusion; (3) ascitic/pericardial fluid, serum, and pleural levels of VEGF are decreased in patients with TB compared with patients with malignancy; (4) pleural levels of VEGF are lower in patients with TPE compared with those with empyema and PPE, whereas serum levels of VEGF are not different between these patients; (5) both CSF and serum levels of VEGF are increased in patients with active TBM compared with controls, including patients with inactive TBM or non-TBM subjects; (6) post-ATT levels of VEGF are increased compared with pre-ATT levels of VEGF; and (7) the mean age and male percentage of the TB group explained large and total amount of heterogeneity for the meta-analysis of blood and pleural VEGF levels compared with healthy controls and patients with PPE, respectively, whereas these moderators did not show any significant interaction with the effect size for other analyses.
The potential role of VEGF in the pathophysiology of TB is derived from different pieces of evidence. Macrophages and their behavior crucially contribute to the determination of host responses against M. tb infection [57]. In vitro studies show that VEGF is among the angiogenic factors that M. tb-infected macrophages secrete to facilitate the spread of M. tb infection from the primary site of infection. e.g., the lung, to a different site [35]. In animal studies, anti-VEGF treatment decreases the spread of M. tb infection [35]. VEGF expression is also found in the brains of animals with TBM, while it was absent in the brains of vaccinated animals [58]. In addition, the observation of lung tissues obtained from patients with fibro-cavernous TB identified the VEGF-A-expressing macrophages distributed over the pyogenic and granulation layers, the areas of fibrosis, draining bronchus, distelectase, and emphysema, and the intact tissues [59]. These lines of evidence lead us to conclude that endothelial function might be altered in TB, and VEGF is crucially involved in TB-related changes in endothelial function.
Clinical studies support the potential use of VEGF as a biomarker of disease severity and activity. Compared with controls with inactive, old, or cured disease, patients with active TB reveal higher VEGF levels [6,7,60]. Also, smear-positive patients and culture-positive patients have higher VEGF levels than their smear-negative and culture-negative counterparts [61]. Among patients with pulmonary TB, the increase of systemic VEGF is more prominent in patients with cavitation [61,62] and in those who have bilateral involvement [62]. For patients with TPE, pleural VEGF levels in patients with loculated pleural effusion are higher than those in patients with non-loculated pleural effusion, and that patients with higher pleural levels of VEGF at baseline appear to be more likely to develop residual pleural thickening (RPE) [63]. In addition, patients with drug-resistant TB with higher systemic levels of VEGF at baseline might be more prone to delays in sputum culture conversion [61].
The development of agents that target VEGF-mediated angiogenesis might help balance endothelial function and add to the value of standard ATT. Bevacizumab is an anti-VEGF drug used for different types of cancers and eye diseases. It has been shown to normalize the vascular dynamics of granuloma, an organized cellular structure that contains immune cells and is recognized as a hallmark of TB, making it more versatile to therapeutic medications [64]. Also, there have been reported cases with ocular TB who responded to anti-VEGF treatments [65,66].
Although the main finding of higher blood VEGF levels in active TB compared with healthy controls is robust as the leave-one-out results revealed, the large amount of heterogeneity remains to be investigated. This meta-analysis is based on 16 observations (N = 1510), from which 13 (81.3%) reported higher VEGF levels in patients, one reported lower VEGF levels in patients, and two reported no difference between patients and control. First, we attempted to do separate latent TB infection and TB-uninfected subjects from a mixed healthy control group. However, it was not possible since most studies (62.5%) did not consider the diagnosis of latent TB infection in healthy controls [5,7,10,17,19,27,34,35,39,41], while some studies addressed the issue and provided the related VEGF data [6,13,31,52]. Second, we performed subgroup analyses by the geographic location, the blood fraction assessed, and the assay used. The effect sizes were significant for specific subgroups of studies (East Asia & Pacific, serum, and ELISA); however, these predictors could not explain the substantial heterogeneity. Finally, both the mean age and male percentage of the TB group were not significant moderators of the SMD and its variance.
Another main finding of our meta-analysis is that both serum and pleural VEGF levels in patients with TPE are increased compared with those with transudate, CHF/CRF, or cirrhotic effusion. CHF is one of the common etiologies of transudate effusion characterized by endothelial dysfunction. Studies suggest that VEGF levels in patients with CHF increase to maintain endothelial repair [67,68]. Again, we could not separate CHF and other types of transudate effusion from the mixed group of transudate effusions. Overall, for blood VEGF levels (k = 7), four observations reported higher VEGF levels in patients with TPE [17,21,26,33], and three found no difference between patients with TPE and patients with transudative, CHF/CFR, or cirrhosis effusion [22,24,41]. For pleural VEGF levels, 13 observations indicated higher VEGF levels in TPE [9, 15, 17, 21-24, 26, 33, 40, 41, 47, 48], while five found no difference [16,20,43,44,49]. There was moderate to high heterogeneity for pleural (I 2 = 71.6%) and blood (I 2 = 80.8%) VEGF levels. The leave-one-out results showed that both effect sizes are robust; however, the study by Fathi and colleagues and the study by Momi and colleagues were found to be accounted for greater than 10% of the true variance. Due to the small number of observations included in the meta-analysis of blood VEGF levels, we could not run subgroup analysis and meta-regression. For pleural VEGF levels, we found that higher VEGF levels in TPE are significant regardless of the geographic location; however, the heterogeneity was lower in Europe & Central Asia studies than those of the Middle East & North Africa (61.9% vs. 88.5%). Meta-regressions did not show any significant association between the effect size and tested moderators.
Our meta-analyses of VEGF levels in TB compared with malignancy were based on 25, 12, and 3 observations for pleural, blood, and ascitic/pericardial fluid samples. The SMD for all analyses was statistically large, calculated as -1.47, -1.30, and -1.54, respectively, and robust as the influence analysis results demonstrated. Several meta-analyses have been published considering the VEGF profile and its prognostic significance in different types of cancer [69][70][71][72]. These studies suggest the hyperactivated VEGF pathway as a target in cancer therapy [73]. Our meta-analyses confirm the previous meta-analyses that VEGF levels are increased in malignancy and add that the increase of VEGF in TB is significantly less than that in malignancy. There was high heterogeneity for both blood and pleural VEGF (I 2 > 90%). We did subgroup analysis by geographic location and observed that lower blood and pleural VEGF levels in TB exist in specific subgroups of studies of East Asia & Pacific (for both blood and pleural levels) and the Middle East and North Africa (for pleural levels). However, again, there was high heterogeneity in these subgroups (I 2 > 90%). Meta-regressions showed that the mean age of patients was accounted for more than 70% of the true variance among studies of blood VGEF levels. However, no such interaction with the effect size was found for the male percentage of patients.
Accordingly, the important limitation of the study is that we could not address the high heterogeneity among studies of blood VEGF in TB vs. healthy controls and of pleural VEGF in TB vs. malignancy. There might be unmeasured factors behind this heterogeneity that need to be explored in future research. Another limitation of the study was that we could not do within-group meta-analyses to compare VEGF levels between the TB subgroups, e.g., pulmonary vs. extrapulmonary TB, TB with cavitation vs. TB without cavitation, TB with RPT vs. TB without RPT, smear-positive TB vs. smear-negative TB, and HIV-infected TB vs. HIV-uninfected TV, since the number of observations was small. Again, for TBM, the most lethal form of TB, we could not investigate the association between the VEGF profile and the disease outcome, given the small number of observations with available data.

Conclusion
This meta-analysis results suggest that blood, CSF, and pleural levels of VEGF are increased in patients with active TB, TPE, and TBM compared with healthy controls, patients with transudative effusions, and non-TBM controls. However, the TB-related increase in ascitic, blood, and pleural VEGF is lower than malignancy-related increase. This calls the need for the development of anti-VEGF treatments that specifically target the TB-related change in VEGF.